Developmental origins of Parkinson's disease risk: perinatal exposure to the organochlorine pesticide dieldrin leads to sex-specific DNA modifications in critical neurodevelopmental pathways in the mouse midbrain.

Epidemiological studies show that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Animal studies support a link between developmental dieldrin exposure and increased neuronal susceptibility in the α-synuclein preformed fibril (α-syn PFF) and MPTP models in adult male C57BL/6 mice. In a previous study, we showed that developmental dieldrin exposure was associated with sex-specific changes in DNA modifications within genes related to dopaminergic neuron development and maintenance at 12 weeks of age. Here, we used capture hybridization-sequencing with custom baits to interrogate DNA modifications across the entire genetic loci of the previously identified genes at multiple time points - birth, 6 weeks, 12 weeks, and 36 weeks old. We identified largely sex-specific dieldrin-induced changes in DNA modifications at each time point that annotated to pathways important for neurodevelopment, potentially related to critical steps in early neurodevelopment, dopaminergic neuron differentiation, synaptogenesis, synaptic plasticity, and glial-neuron interactions. Despite large numbers of age-specific DNA modifications, longitudinal analysis identified a small number of DMCs with dieldrin-induced deflection of epigenetic aging. The sex-specificity of these results adds to evidence that sex-specific responses to PD-related exposures may underly sex-specific differences in disease. Overall, these data support the idea that developmental dieldrin exposure leads to changes in epigenetic patterns that persist after the exposure period and disrupt critical neurodevelopmental pathways, thereby impacting risk of late life diseases, including PD.

Alpha-synuclein inclusion responsive microglia are resistant to CSF1R inhibition.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously determined that microglia proximal to PFF-induced nigral α-syn inclusions increase in soma size, upregulate major-histocompatibility complex-II (MHC-II) expression, and increase expression of a suite of inflammation-associated transcripts. This microglial response is observed months prior to degeneration, suggesting that microglia reacting to α-syn inclusion may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether colony stimulating factor-1 receptor (CSF1R)-mediated microglial depletion impacts the magnitude of α-syn aggregation, nigrostriatal degeneration, or the response of microglial in the context of the α-syn PFF model. METHODS: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600 mg/kg), a CSF1R inhibitor, to deplete microglia for a period of either 2 or 6 months. RESULTS: CSF1R inhibition resulted in significant depletion (~ 43%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. However, CSF1R inhibition did not impact the increase in microglial number, soma size, number of MHC-II immunoreactive microglia or microglial expression of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, and Fcer1g associated with phosphorylated α-syn (pSyn) nigral inclusions. Further, accumulation of pSyn and degeneration of nigral neurons was not impacted by CSF1R inhibition. Paradoxically, long term CSF1R inhibition resulted in increased soma size of remaining Iba-1ir microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. CONCLUSIONS: Collectively, our results suggest that CSF1R inhibition does not impact the microglial response to nigral pSyn inclusions and that CSF1R inhibition is not a viable disease-modifying strategy for PD.

Deficits in basal and evoked striatal dopamine release following alpha-synuclein preformed fibril injection: An in vivo microdialysis study.

Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% âž” 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.

Transcriptomic profiling of early synucleinopathy in rats induced with preformed fibrils.

Examination of early phases of synucleinopathy when inclusions are present, but long before neurodegeneration occurs, is critical to both understanding disease progression and the development of disease modifying therapies. The rat alpha-synuclein (α-syn) preformed fibril (PFF) model induces synchronized synucleinopathy that recapitulates the pathological features of Parkinson's disease (PD) and can be used to study synucleinopathy progression. In this model, phosphorylated α-syn (pSyn) inclusion-containing neurons and reactive microglia (major histocompatibility complex-II immunoreactive) peak in the substantia nigra pars compacta (SNpc) months before appreciable neurodegeneration. However, it remains unclear which specific genes are driving these phenotypic changes. To identify transcriptional changes associated with early synucleinopathy, we used laser capture microdissection of the SNpc paired with RNA sequencing (RNASeq). Precision collection of the SNpc allowed for the assessment of differential transcript expression in the nigral dopamine neurons and proximal glia. Transcripts upregulated in early synucleinopathy were mainly associated with an immune response, whereas transcripts downregulated were associated with neurotransmission and the dopamine pathway. A subset of 29 transcripts associated with neurotransmission/vesicular release and the dopamine pathway were verified in a separate cohort of males and females to confirm reproducibility. Within this subset, fluorescent in situ hybridization (FISH) was used to localize decreases in the Syt1 and Slc6a3 transcripts to pSyn inclusion-containing neurons. Identification of transcriptional changes in early synucleinopathy provides insight into the molecular mechanisms driving neurodegeneration.

Developmental exposure to the Parkinson's disease-associated organochlorine pesticide dieldrin alters dopamine neurotransmission in α-synuclein pre-formed fibril (PFF)-injected mice.

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration.

Microglial depletion does not impact alpha-synuclein aggregation or nigrostriatal degeneration in the rat preformed fibril model.

Background: Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously described the time course of microglial major-histocompatibility complex-II (MHC-II) expression and alterations in microglia morphology in the PFF model in rats. Specifically, the peaks of α-syn inclusion formation, MHC-II expression, and reactive morphology in the substantia nigra pars compacta (SNpc) all occur two months post PFF injection, months before neurodegeneration occurs. These results suggest that activated microglia may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether microglial depletion could impact the magnitude of α-syn aggregation, nigrostriatal degeneration, or related microglial activation during the α-syn PFF model. Methods: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600mg/kg), a colony stimulating factor-1 receptor (CSF1R) inhibitor, to deplete microglia for a period of either two or six months. Results: PLX3397B administration resulted in significant depletion (45-53%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. Microglial depletion did not impact accumulation of phosphorylated α-syn (pSyn) within SNpc neurons and did not alter pSyn associated microglial reactivity or expression of MHC-II. Further, microglial depletion did not impact SNpc neuron degeneration. Paradoxically, long term microglial depletion resulted in increased soma size of remaining microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. Conclusions: Collectively, our results suggest that microglial depletion is not a viable disease-modifying strategy for PD and that partial microglial depletion can induce a heightened proinflammatory state in remaining microglia.

Parkinson's disease-associated, sex-specific changes in DNA methylation at PARK7 (DJ-1), SLC17A6 (VGLUT2), PTPRN2 (IA-2ß), and NR4A2 (NURR1) in cortical neurons.

Evidence for epigenetic regulation playing a role in Parkinson's disease (PD) is growing, particularly for DNA methylation. Approximately 90% of PD cases are due to a complex interaction between age, genes, and environmental factors, and epigenetic marks are thought to mediate the relationship between aging, genetics, the environment, and disease risk. To date, there are a small number of published genome-wide studies of DNA methylation in PD, but none accounted for cell type or sex in their analyses. Given the heterogeneity of bulk brain tissue samples and known sex differences in PD risk, progression, and severity, these are critical variables to account for. In this genome-wide analysis of DNA methylation in an enriched neuronal population from PD postmortem parietal cortex, we report sex-specific PD-associated methylation changes in PARK7 (DJ-1), SLC17A6 (VGLUT2), PTPRN2 (IA-2ß), NR4A2 (NURR1), and other genes involved in developmental pathways, neurotransmitter packaging and release, and axon and neuron projection guidance.

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

Dopaminergic Positron Emission Tomography Imaging in the Alpha-Synuclein Preformed Fibril Model Reveals Similarities to Early Parkinson's Disease.

BACKGROUND: Positron emission tomography (PET) imaging in early Parkinson's disease (PD) subjects reveals that increased dopamine (DA) turnover and reduced dopamine transporter (DAT) density precede decreases in DA synthesis and storage. The rat α-synuclein preformed fibril (α-syn PFF) model provides a platform to investigate DA dynamics during multiple stages of α-syn inclusion-triggered nigrostriatal degeneration. OBJECTIVES: We investigated multiple aspects of in vivo dopaminergic deficits longitudinally and similarities to human PD using translational PET imaging readouts. METHODS: Longitudinal imaging was performed every 2 months in PFF and control rats for 7 months. [18 F]-Fluoro-3,4-dihydroxyphenyl-L-alanine (FDOPA) imaging was performed to investigate DA synthesis and storage (Kocc ) and DA turnover, estimated by its inverse, the effective distribution volume ratio (EDVR). 11 C-Methylphenidate (MP) was used to estimate DAT density (BPND ). RESULTS: Early DA turnover increases and DAT binding decreases were observed in the ipsilateral striatum of PFF rats, progressing longitudinally. EDVR decreased 26%, 38%, and 47%, and BPND decreased 36%, 50%, and 65% at the 2-, 4-, and 6-month time points, respectively, compared to ipsilateral control striatum. In contrast, deficits in DA synthesis and storage were not observed in the ipsilateral striatum of PFF rats compared to control injections and were relatively preserved up to 6 months (Kocc decreased 20% at 6 months). CONCLUSIONS: The relative preservation of DA synthesis and storage compared to robust progressive deficits in DAT density and increases in DA turnover in the rat α-syn PFF model display remarkable face validity to dopaminergic alterations in human PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease.

The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson's disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.

Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits.

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.

Basic Concepts in Viral Vector-Mediated Gene Therapy.

Today any researcher with the desire can easily purchase a viral vector. However, despite the availability of viral vectors themselves, the requisite knowledge that is absolutely essential to conducting a gene therapy experiment remains somewhat obscure and esoteric. To utilize viral vectors to their full potential, a large number of decisions must be made, in some instances prior to even obtaining the vector itself. For example, critical decisions include selection of the proper virus, selection of the proper expression cassette, whether to produce or purchase a viral vector, proper viral handling and storage, the most appropriate delivery method, selecting the proper controls, how to ensure your virus is expressing properly, and many other complex decisions that are essential to performing a successful gene therapy experiment. The need to make so many important decisions can be overwhelming and potentially prohibitive, especially to the novice gene therapist. In order to aid in this challenging process, here we provide an overview of basic gene therapy modalities and a decision tree that can be used to make oneself aware of the options available to the beginning gene therapist. This information can be used as a road map to help navigate the complex and perhaps confusing process of designing a successful gene therapy experiment.

Multimodal Production of Adeno-Associated Virus.

Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer.

Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology.

Alpha-Synuclein (α-syn) is by far the most highly vetted pathogenic and therapeutic target in Parkinson's disease. Aggregated α-syn is present in sporadic Parkinson's disease, both in the central nervous system (CNS) and peripheral nervous system (PNS). The enteric division of the PNS is of particular interest because 1) gastric dysfunction is a key clinical manifestation of Parkinson's disease, and 2) Lewy pathology in myenteric and submucosal neurons of the enteric nervous system (ENS) has been referred to as stage zero in the Braak pathological staging of Parkinson's disease. The presence of Lewy pathology in the ENS and the fact that patients often experience enteric dysfunction before the onset of motor symptoms has led to the hypothesis that α-syn pathology starts in the periphery, after which it spreads to the CNS via interconnected neural pathways. Here we sought to directly test this hypothesis in rodents and non-human primates (NHP) using two distinct models of α-syn pathology: the α-syn viral overexpression model and the preformed fibril (PFF) model. Subjects (rat and NHP) received targeted enteric injections of PFFs or adeno-associated virus overexpressing the Parkinson's disease associated A53T α-syn mutant. Rats were evaluated for colonic motility monthly and sacrificed at 1, 6, or 12 months, whereas NHPs were sacrificed 12 months following inoculation, after which the time course and spread of pathology was examined in all animals. Rats exhibited a transient GI phenotype that resolved after four months. Minor α-syn pathology was observed in the brainstem (dorsal motor nucleus of the vagus and locus coeruleus) 1 month after PFF injections; however, no pathology was observed at later time points (nor in saline or monomer treated animals). Similarly, a histopathological analysis of the NHP brains revealed no pathology despite the presence of robust α-syn pathology throughout the ENS which persisted for the entirety of the study (12 months). Our study shows that induction of α-syn pathology in the ENS is sufficient to induce GI dysfunction. Moreover, our data suggest that sustained spread of α-syn pathology from the periphery to the CNS and subsequent propagation is a rare event, and that the presence of enteric α-syn pathology and dysfunction may represent an epiphenomenon.

Targeted gene delivery to the enteric nervous system using AAV: a comparison across serotypes and capsid mutants.

Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm(2). AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS.

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain.

Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson's disease. Although viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be because of the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing Parkinson's disease and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared with the young adult. We examined recombinant adeno-associated virus 2/5-mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra aged rats displayed 40%-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Furthermore, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP messenger RNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less messenger RNA and protein expression.

Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system.

The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson's disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 10(13)) or varying titers (2.2 x 10(12), 1.0 x 10(13), 5.9 x 10(13), or 1.0 x 10(14)) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 10(14) titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 10(13) titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 10(13) and 1.0 x 10(14) rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 10(13) rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.